Hydrogels and hydrogel arrays made from reactive prepolymers crosslinked by [2 + 2] cycloaddition

ABSTRACT

Reactive prepolymers incorporating [2+2] photoreactive sites are synthesized. Upon exposure to UV light, these prepolymers undergo [2+2] cycloaddition to crosslink. When crosslinked, the reactive prepolymers form a hydrogel. Selective hydrogel formation is provided through selective exposure of the reactive prepolymer to UV light. Supports and other molecules may be attached or incorporated into the hydrogel through [2+2] cycloaddition with uncrosslinked [2+2] photoreactive sites present in the hydrogel.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Nonprovisional Application No. 09/344,217, filed Jun. 25, 1999, entitled “Polyacrylamide Hydrogels and Hydrogel Arrays Made from Polyacrylamide Reactive Prepolymers,” which claimed the benefit of U.S. Provisional Application No. 60/109,821, filed Nov. 25, 1998 entitled “Polyacrylamide Hydrogels and Hydrogel Arrays Made from Polyacrylamide Reactive Prepolymers.”

BACKGROUND

[0002] Acrylamide (CH₂═CHC(O)NH₂; C.A.S. 79-06-1; also known as acrylamide monomer, acrylic amide, propenamide, and 2-propenamide) is an odorless, free-flowing white crystalline substance that may be polymerized to form polyacrylamides. The resulting high molecular weight polymers have a variety of uses and further can be modified to optimize nonionic, anionic, or cationic properties for specified uses.

[0003] Polyacrylamide hydrogels are used as molecular sieves for the separation of nucleic acids, proteins, peptides, oligonucleotides, polynucleotides, and other biological materials. They are also used as binding layers for biological materials. When used as binding layers, the gels currently are produced as thin sheets or slabs, typically by depositing a solution of acrylamide monomer, a crosslinker such methylene bisacrylamide, and an initiator such as N,N,N′,N′-tetramethylethylendiamine (TEMED) between two glass surfaces (e.g., glass plates or microscope slides) using a spacer to obtain the desired thickness of polyacrylamide.

[0004] Generally, the acrylamide polymerization solution is a 4-5% solution (acrylamide/bisacrylamide 19/1) in water/glycerol, with a nominal amount of initiator. The acrylamide is chain-polymerized and crosslinked by radical initiation from ultraviolet (UV) radiation (e.g., 254 nm for about 15 minutes) or heat (e.g., about 400° C.). Following polymerization and crosslinking, the top glass slide is removed from the surface to uncover the gel. Changing the amount of crosslinker and the % solids in the monomer solution controls the pore size of the gel. Changing the polymerization temperature also can control the pore size.

[0005] The current approach of making polyacrylamide hydrogels starting from acrylamide monomer has several disadvantages, some of which are described below:

[0006] 1. The coating process is difficult and expensive to automate because the film thickness is controlled with a spacer and top glass plate. The removal of the top glass plate must be done manually. Due to difficulties automating the process (e.g., viscosity too low for commercial coating methods), the coating of the monomer solution currently is done manually.

[0007] 2. The reaction time of the acrylamide is excessively long (e.g., typically from about 15 to about 90 minutes at a short wavelength of about 254 nm), making the UV polymerization and crosslinking step incompatible with standard imaging equipment such as mask aligners and photoprinters.

[0008] 3. The acrylamide monomer is a neurotoxin and a carcinogen which makes coating, handling, and waste disposal of the material hazardous and expensive.

[0009] Added to these disadvantages are the further problems that crystallization of monomer frequently occurs on commonly used equipment and laboratory surfaces, and exothermic polymerization can occur in coating reservoirs, necessitating the use of stabilizers or inhibitors. The present invention overcomes at least one of these disadvantages.

BRIEF SUMMARY

[0010] Polyacrylamide hydrogels that incorporate [2+2] photoreactive sites are disclosed. The hydrogels are especially useful for microarray formation and are made from prepolymers, including polyacrylamide reactive prepolymers. The photoreactive sites allow use of [2+2] cycloaddition reactions to not only crosslink the polyacrylamide reactive prepolymers forming the hydrogel, but also for later attachment of any other molecules incorporating additional photoreactive sites. The disclosed hydrogels provide a more uniform pore size, likely resulting from an improved control of crosslinking, making them preferable for use with DNA based probes. Additionally, because higher viscosity prepolymers, as opposed to low viscosity monomer solutions, are used to form the arrays, manufacturing is simplified.

[0011] The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 depicts a DMI based polyacrylamide reactive prepolymer formed by a “one-step” process. The polyacrylamide reactive prepolymer (PRP) was formed by thermally polymerizing monomers of acrylamide and N-(6-acryloylhexyl)-2,3-dimethylmaleimide. Because N-(6-acryloylhexyl)-2,3-dimethylmaleimide is “bifunctional,” (having both a polymerizable functionality and a [2+2] photoreactive site in a single molecule) it forms a PRP directly upon copolymerization with acrylamide. Upon irradiation with ultraviolet light, the PRP is crosslinked to form a hydrogel by cycloaddition at the [2+2] photoreactive sites. Substituents x and y can be varied between 1 and 50. The wavy line (~~~~~) between oxygen and nitrogen represents the hexyl spacer group. The dashed bonds (———) indicate attachment points for additional monomers.

[0013]FIG. 2 depicts a “two-step” reaction scheme for reactive prepolymer formation. First, a copolymer of acrylamide and acrylic acid is formed. Second, the copolymer is treated with a [2+2] photoreactive compound, such as acryloyl chloride. The acrylic acid monomer then condenses with the acryloyl chloride to give a PRP with an acrylate provided [2+2] photoreactive site. Substituents x and y can be varied between 1 and 50. The dashed bonds indicate attachment points for additional monomers.

[0014]FIG. 3 depicts a Glycidyl Methacrylate Based PRP resulting from a two-step process in which a copolymer of acrylamide and acrylic acid is reacted with glycidyl methacrylate. An acrylate provided [2+2] photoreactive site results. Because [2+2] photoreactive sites provided by acrylate groups will not photocyclize with themselves, but will undergo undesirable chain-type polymerization if irradiated with UV light, a mixed hydrogel is formed by ultraviolet exposure in the presence of a DMI based PRP. Alternatively, an additional crosslinking agent can be used to form a hydrogel from the acrylate based PRP. Substituents x and y can be varied between 1 and 50. The dashed bonds indicate attachment points for additional monomers.

DETAILED DESCRIPTION

[0015] Overview

[0016] Hydrogels suitable for forming microarrays and continuous films to which probes may be attached by [2+2] photocycloaddition chemistry are described. The arrays or films are formed by first synthesizing a reactive prepolymer that incorporates [2+2] photocyclizable sites. A thin layer of prepolymer solution is then placed on a solid support and exposed to ultraviolet light. If microarrays are desired, the hydrogel layer is selectively exposed to ultraviolet light. During exposure, a portion of the [2+2] photocyclizable sites exposed to the light undergoes cyclization to crosslink the reactive prepolymer, thus forming a hydrogel. The uncyclized reactive prepolymer is then washed away with an aqueous solution. Unlike the reactive prepolymer, the crosslinked hydrogel is not water-soluble. A continuous hydrogel film or microarray of hydrogel sites attached to the solid support results.

[0017] Probes or other molecules containing [2+2] photoreactive sites may undergo cycloaddition with the hydrogel through further UV exposure. Sufficient [2+2] photoreactive sites are incorporated into the reactive prepolymer to crosslink the polymer to form the hydrogel and to remain available for later probe and other molecule attachment.

[0018] For arrays, the hydrogel pattern results from selective irradiation with ultraviolet light. Selective irradiation may be accomplished in multiple ways known to those of skill in the art. Preferable methods include (1) using an opaque mask to shield portions of the reactive prepolymer from ultraviolet exposure and (2) using a laser beam to only irradiate selected positions on the reactive prepolymer. Either method results in only selected portions of the reactive polymer being crosslinked. The uncrosslinked reactive polymer can then be removed to leave the array grid on the support.

[0019] Reactive Prepolymer

[0020] While a variety of compounds or molecules may be used to form the reactive prepolymer, two basic procedures are preferred. In the first, a bifunctional molecule with a polymerizable end and a [2+2] photocyclizable end is copolymerized with a hydrophilic first monomer. In the second, a second monomer that may be attached to a compound having a [2+2] photoreactive site is copolymerized with the hydrophilic first monomer. This copolymer is then reacted with a compound having a [2+2] photoreactive site to produce the desired reactive prepolymer. Either method results in a reactive prepolymer that may be crosslinked into a hydrogel by [2+2] photocycloaddition.

[0021] Monomers

[0022] [0019] Monomers are the individual molecular units that are repeated to form polymers. Multiple monomers covalently attached form the backbone of a polymer. Polymers that are made from at least two different monomer units are referred to as copolymers. Polymerizing or copolymerizing describes the process by which multiple monomers are covalently linked to form polymers or copolymers, respectively. A discussion of polymers and monomers from which they are made may be found in Stevens, Polymer Chemistry: An Introduction, 3^(rd) ed., Oxford University Press, 1999. The preferred embodiments rely on three general classes of monomers to form reactive prepolymers.

[0023] The monomers used in the preferred embodiments may be covalently attached by chain-type polymerization chemistry to form the backbone of the reactive prepolymer. Thus, these monomers are “polymerizable.”

[0024] Chain-type Polymerization

[0025] Chain-type polymerization is a polymerization reaction that is radical initiated. An energetic radical is formed in the presence of the monomers, which then join to form a polymer. Many methods of radical initiation are known, but most rely on thermal or light energy to generate the reactive radicals. In one embodiment, thermal radical initiation is most preferred to form a polymer backbone incorporating acrylamide first monomers.

[0026] If the radicals are light generated, preferred initiators include N,N,N′, N′-tetramethylethylendiamine (TEMED) and benzophenones. Preferable thermal initiators include peroxides, such as benzoyl peroxide. A chain-type polymerization reaction is depicted below.

[0027] In order to undergo chain-type polymerization, a monomer must have “polymerizable functionality,” as provided by the olefins in the butylenes depicted above. During this butylene polymerization, for example, the unsaturated monomers covalently bond to form a saturated polybutylene polymer. The monomers are covalently attached through their polymerizable groups.

[0028] First Monomers

[0029] The first type of monomer, or “first monomer,” is preferably water-soluble and polymerizable. First monomers may be copolymerized by chain-type methods with other polymerizable monomers and impart hydrophilicity to the resultant copolymer. After polymerization, the first monomers are covalently bonded to themselves or other polymerizable monomers. They are also resistant to [2+2] cyclization reactions as described below. By cyclization resistant, it is meant that when heated or exposed to ultraviolet light, first monomers will preferentially undergo chain-type polymerization in relation to [2+2] cyclization.

[0030] Monomers that undergo chain-type radical polymerization, resist [2+2] photocyclization, and solubilize in water are preferred first monomers. Examples of preferred first monomers include acrylamide, hydroxyethyl acrylate, vinyl pyridine, acrylic acid, methacrylic acid, and vinyl pyrrolidone, or mixtures thereof. Most preferred is acrylamide.

[0031] Second Monomer

[0032] The second type of monomer, or “second monomer,” may be copolymerized by chain-type methods with other polymerizable monomers and includes a heteroatom (e.g. oxygen, nitrogen, sulfur, phosphorous). After copolymerization, the heteroatom may be used to covalently attach the second monomer to a [2+2] photocyclizable compound by a condensation reaction.

[0033] A condensation reaction occurs when two molecules are covalently joined through a non-radical pathway. The condensation of acrylic acid with glycidyl methacrylate is shown below. In this reaction the oxygen heteroatom of acrylic acid opens the epoxide ring of glycidyl acrylate to form a covalent bond.

[0034] Preferred second monomers can be any monomer that is polymerizable and has a heteroatom suitable for bonding with a compound having a [2+2] photoreactive site, as described below. More preferred second monomers include acrylic acid, glycidyl methacrylate, and methacrylic acid, or mixtures thereof. Acrylic acid is most preferred.

[0035] Preferable [2+2] photocyclizable compounds include any compound incorporating a [2+2] photoreactive site that can undergo a condensation reaction with a second monomer. More preferred [2+2] photocyclizable compounds include glycidyl methacrylate, acrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, and acryloyl halides, or mixtures thereof.

[0036] Bifunctional Monomers

[0037] The third type of monomers used to form reactive prepolymers includes both polymerizable functionality and [2+2] photoreactive sites in a single monomer. By tuning the electron density and steric parameters of a monomer to favor polymerization (greater electron density, less sterics) at one end and to favor [2+2] cycloaddition (less electron density, greater sterics) at the other, bifunctional monomers may be formed. When copolymerized with first monomers, bifunctional monomers form reactive prepolymers without requiring further incorporation of [2+2] photocyclizable compounds. Preferably, bifunctional monomers are thermally copolymerized with a first monomer and then exposed to ultraviolet light to crosslink a portion of the [2+2] photoreactive sites to form a hydrogel.

[0038] Preferable bifunctional monomers incorporate vinyl and dimethylmaleimide or cinnamate groups. Most preferred are N-(6-acryloylhexyl)-2,3-dimethylmaleimide (as shown below) and vinyl cinnamate.

[0039] [2+2] Photocyclizable Monomer

[0040] [0032] [2+2] photoreactive sites can undergo [2+2] photocycloaddition and are therefore [2+2] photocyclizable. [2+2] photocyclizable monomers are preferably any monomer unit that includes a [2+2] photoreactive site and a polymerizable site. Therefore, [2+2] photocyclizable monomers can be either bifunctional, like DMI, or made through condensation of a polymerizable compound with a [2+2] photocyclizable compound.

[0041] A monomer or compound incorporating a [2+2] photoreactive site can undergo [2+2] cycloaddition with other monomers or compounds that possess a [2+2] photoreactive site. If a monomer or compound incorporating a [2+2] photoreactive site is irradiated with ultraviolet light, the [2+2] reactive portion of the molecule will preferentially undergo cycloaddition or “cyclize” with other [2+2] photoreactive sites, instead of undergoing chain-type polymerization. Preferably, polymerizable sites have a greater electron density and are less sterically hindered than [2+2] photoreactive sites.

[0042] While second monomers and [2+2] photocyclizable compounds serve different chemical functions in the present embodiments, some molecules can provide either functionality depending on reaction order. For example, if acrylamide is copolymerized with acrylic acid and then condensed with glycidyl methacrylate, acrylic acid provides second monomer functionality and glycidyl methacrylate provides [2+2] photocyclizable compound functionality. Alternatively, if acrylamide is copolymerized with glycidyl methacrylate and then condensed with acrylic acid, glycidyl methacrylate provides second monomer functionality and acrylic acid provides [2+2] photocyclizable compound functionality. In either reaction sequence, a PRP is formed with an acrylate based [2+2] photoreactive site. This reversibility holds true for the reaction of acrylic acid with glycidyl methacrylate, hydroxyethyl acrylate, or hydroxypropyl acrylate. Hence, reaction order can determine whether a compound serves as a second monomer or a [2+2] photoreactive compound during two-step reactive prepolymer synthesis.

[0043] [2+21] Cyclization

[0044] In the disclosed embodiments, [2+2] cyclization or cycloaddition is a light-induced reaction between two photoreactive sites, at least one of which is electronically excited. Advantageously, [2+2] cycloaddition reactions can proceed with high efficiency. While it is chemical convention to write cycloaddition centers in brackets, such as “[2+2]” or “[4+2],” the brackets were omitted from the claims to prevent confusion with the patent convention of deleting bracketed material. Hence, in the claims “[2+2]” is written as “2+2.”

[0045] Most preferably, cyclization is of the [2+2] variety, wherein two carbon-carbon or a carbon-carbon and a carbon-heteroatom single bond are formed in a single step. The [2+2] cycloaddition involves addition of a 2π-component of a double bond to the 2π-component of a second double bond, as shown below.

[0046] Alternatively, the reaction may proceed by way of a 2π-component of triple bonds. Under the rules of orbital symmetry, such [2+2] cycloadditions are thermally forbidden, but photochemically allowed. Such reactions typically proceed with a high degree of stereospecificity and regiospecificity.

[0047] Photochemical [2+2] cycloaddition of monomers on opposing copolymer chains results in crosslinking of the copolymer backbones. In addition to cyclizing with other photocyclizable monomers, [2+2] photocyclizable monomers can cyclize with other molecules, such as DNA probes, which incorporate [2+2] photoreactive sites. In this manner, other molecules may be attached to the preferred hydrogels. The additional [2+2] cyclizable molecules may be cyclized with the monomers during or after the cyclization reaction that crosslinks the monomers to form the hydrogel. The cyclization between monomers results in covalent crosslinking to form hydrogels, while cyclization between monomers and other molecules results in covalent attachment of the other molecules to the hydrogel. In a likewise fashion, the reactive prepolymer may be cyclized to a solid support, such as glass or plastic, which incorporates [2+2] photoreactive sites.

[0048] Preferred [2+2] cycloadditions include those between two carbon-carbon double bonds to form cyclobutanes and those between alkenes and carbonyl groups to form oxetanes. Cycloadditions between two alkenes to form cyclobutanes can be carried out by photo-sensitization with mercury or directly with short wavelength light, as described in Yamazaki et al., J. Am. Chem. Soc., 91, 520 (1969). The reaction works particularly well with electron-deficient double bonds because electron-poor olefins are less likely to undergo undesirable side reactions. Cycloadditions between carbon-carbon and carbon-oxygen double bonds, such as α,β-unsaturated ketones, form oxetanes (Weeden, In Synthetic Organic Photochemistry, Chapter 2, W. M. Hoorspool (ed.) Plenum, New York, 1984) and enone addition to alkynes (Cargill et al., J. Org. Chem., 36, 1423 (1971)).

[0049] Photoreactive Sites

[0050] Photoreactive sites are defined as chemical bonds capable of undergoing [2+2] cycloaddition (cyclization) to form a ring structure when exposed to light of an appropriate wavelength. Photoreactive sites can yield homologous linking, where a monomer or other molecule photoreactive site cyclizes with a monomer or other molecule photoreactive site having the same chemical structure, or for heterologous linking, where a monomer or other molecule photoreactive site cyclizes with a monomer or other molecule photoreactive site having a different chemical structure.

[0051] In the current embodiments, DMI and vinyl cinnamate [2+2] photoreactive sites can cyclize with themselves, each other, or acrylate provided [2+2] photoreactive sites. Unlike the photoreactive sites of DMI and vinyl cinnamate, however, acrylate provided [2+2] photoreactive sites do not undergo [2+2] photocycloaddition with themselves. Preferred homologous linking occurs between dimethyl maleimide (DMI) photoreactive sites, while preferred heterologous linking occurs between acrylate and DMI photoreactive sites. A detailed discussion of photoreactive sites may be found in Guillet, Polymer Photophysics and Photochemistry, Ch. 12 (Cambridge University Press: Cambridge, London). Generally, double bonds that are not part of a highly conjugated system (e.g. benzene will not work) are preferred. Sterically-hindered, electron deficient double bonds, such as found in maleimide, are most preferred.

[0052] Additionally, molecules having a structure similar to dimethyl maleimide may be used as photoreactive sites, including maleimide/N-hydroxysuccinimide (NHS) ester derivatives. Such preferred maleimide/NHS esters include 3-maleimidoproprionic acid hydroxysuccinimide ester; 3-maleimidobenzoic acid N-hydroxy succinimide; N-succinimidyl 4-malimidobutyrate; N-succinimidyl 6-maleimidocaproate; N-succinimidyl 8-maleimidocaprylate; N-succinimidyl 11-maleimidoundecaoate. These esters can be obtained from a variety of commercial vendors, such as ALDRICH (Milwaukee, Wis).

[0053] Ultraviolet Irradiation

[0054] Crosslinking of the [2+2] photoreactive sites of the photocyclizable monomers in the reactive prepolymer is most preferably done with ultraviolet irradiation. Optionally, a photosensitiser may be added to the reactive prepolymer to increase the efficiency of the cycloaddition reaction. Preferred photosensitisers include water-soluble quinones and xanthones, including anthroquinone, thioxanthone, sulfonic acid quinone, benzoin ethers, acetophenones, benzoyl oximes, acylphosphines, benzophenones, and TEMED (N,N,N′,N′-tetramethylethylendiamine). Anthroquinone-2-sulfonic acid is most preferred and is available from ALDRICH, Milwaukee, Wis.

[0055] While irradiation by light in the ultraviolet spectrum between 250 and 450 nanometers is preferred, longer wavelength ultraviolet radiation is more preferred. Light of about 365 nanometers in wavelength is most preferred. By using longer wavelength ultraviolet light, possible damage of other molecules, such as DNA probes that degrade at 256 nanometers, is avoided. Irradiation is preferably carried out between 22 and 300° C.

[0056] The reactive prepolymer is irradiated with ultraviolet light for preferably between 10 and 100 seconds, most preferably for about 30 seconds. Longer irradiation times result in thicker hydrogels, while shorter irradiation times yield thinner hydrogels. Useful hydrogels are preferably from about 2 nanometers to about 5 micrometers in thickness, more preferably from about 2 nanometers to about 100 nanometers in thickness, and most preferably from about 2 nanometers to about 50 nanometers in thickness. Thickness is defined as the dimension perpendicular to the support.

[0057] While irradiation may be carried out with any device capable of producing light at the preferred wavelengths, a photolithography tool, such as a metal halide lamp equipped Calibre ORC, available from Mentor Graphics (Wilsonville, Oreg.), is most preferred.

[0058] Microarray Formation

[0059] To form a microarray, it is preferable to selectively crosslink only a portion of the reactive prepolymer present on the solid support, thus forming a pattern or grid of separate hydrogel locations. One method of accomplishing this goal is after application of the reactive prepolymer to the support, the reactive prepolymer is covered with a mask having only a portion of its surface transparent to the irradiation light. When the mask is then irradiated, only the portion exposed to the light crosslinks to form a hydrogel. The uncrosslinked reactive polymer is then removed, preferably with aqueous solutions. In this fashion, the pattern of the mask is transferred to the hydrogel microarray. See Sze, VLSI Technology, McGraw-Hill (1983).

[0060] Patterned hydrogel microarrays can also be formed on the support by using an ultraviolet laser to only irradiate the portions of the reactive prepolymer where a hydrogel is desired. See U.S. Pat. No. 4,719,615. After this targeted irradiation, the excess reactive polymer is removed, leaving a microarray of individual hydrogel locations. For either method, the hydrogel locations may preferably be arranged in rows and columns.

[0061] Preferably each location of hydrogel extends from about 2 nanometers to about 5 microns, more preferably from about 2 nanometers to about 100 nanometers, and most preferably from about 2 nanometers to about 50 nanometers in the direction perpendicular to the solid support. Each hydrogel location is preferably about 200 micrometers in diameter, more preferably about 100 micrometers in diameter, and most preferably about 50 micrometers in diameter. Diameter is defined as the dimension parallel to the support.

[0062] Spacers

[0063] Spacers are preferably groups that neither polymerize nor undergo [2+2] cyclization during reactive prepolymer and hydrogel formation and separate the polymerizable and [2+2] photoreactive sites of bifunctional monomers. Spacers may also be used to physically separate second monomers from [2+2] photoreactive sites before condensation of the second monomer with the [2+2] photocyclizable compound. More preferred spacers are —CH₂)_(n)— (methylene) or —(OCH₂CH₂)_(n)— (ethylene oxide), most preferably where n=1-10. A—(CH₂)₆— spacer is present in N-(6-acryloylhexyl)-2,3-dimethylmaleimide, as shown above.

[0064] Reactive Prepolymers

[0065] Reactive prepolymers are water-soluble copolymers that incorporate [2+2] photoreactive sites that are available for [2+2] cycloaddition type crosslinking. Water-solubility is measured by determining the clarity of an aqueous solution containing a specific weight percent of the reactive prepolymer. Preferable reactive prepolymer solutions are clear and contain from about 0.5% to about 22% reactive prepolymer, on a weight basis. The clarity of the reactive prepolymer when coated on the solid support is also a measure of solubility, since if precipitation occurs and water-solubility is lost, an opaque or non-uniform film results. Preferable water-soluble reactive prepolymers form a clear solution when an aqueous solution contains about 22% by weight, or less, of the reactive prepolymer. The aqueous solution to which the reactive prepolymer is added is defined as including at least 80% water by weight.

[0066] Reactive prepolymers preferably have a weight average molecular weight of from about 1,000 to about 300,000 g/mole, more preferably about 5,000 to about 100,000 g/mole, and most preferably about 5,000 to about 50,000 g/mole. However, reactive prepolymers also can be modified from the structures described with other molecules that do not interfere with copolymerization or [2+2] cyclization.

[0067] Generally, if the reactive prepolymer's weight average molecular weight is less than about 1,000 g/mole it can become too brittle to properly coat the solid support. Alternatively, if the weight average molecular weight exceeds about 300,000 g/mole, the reactive prepolymer can become too thick to coat the solid support. Thus, the reactive prepolymer's viscosity is preferably from about 25 centiPoise to about 500,000 centiPoise, more preferably from about 50 centiPoise to about 500,000 centipoise, and most preferably about 200 centiPoise, as measured in deionized water at about 270° C.

[0068] The reactive prepolymer preferably includes between 1 and about 50 first monomers to each bifunctional monomer or second monomer condensed with a [2+2] photocyclizable compound. More preferably, the reactive prepolymer includes between about 10 and about 20 first monomers to each bifunctional monomer or second monomer condensed with a [2+2] photocyclizable compound. Most preferably, the reactive prepolymer includes about 15 first monomers to each bifunctional monomer or second monomer condensed with a [2+2] photocyclizable compound. By varying the amount of first monomer to the bifunctional or second monomer, the number of sites available for [2+2] cyclization may be optimized.

[0069] A preferred reactive prepolymer has the structure:

[0070] where x is an integer from 1 to 50, y is an integer from 1 to 50, and R is a moiety that comprises a 2+2 photoreactive site.

[0071] Hydrogels

[0072] Hydrogels are formed from reactive prepolymers that have had at least a portion of their [2+2] photoreactive sites photochemically crosslinked. While hydrogels are hydrophilic and tend to entrap water, they are not appreciably soluble in water. Preferably, reactive prepolymers that incorporate water-soluble monomers and monomers incorporating [2+2] photoreactive sites are crosslinked to form hydrogels. Generally, homologous hydrogels can be formed from DMI or vinyl cinnamate and heterologous hydrogels can be formed from DMI and vinyl cinnamate, DMI and an acrylate, or vinyl cinnamate and an acrylate. While these molecular pairings are preferred, any alternative compounds that provide the required functionality for polymerization and [2+2] cyclization may be used.

[0073] While not necessary, hydrogels may also be formed by adding an additional crosslinking agent to the reactive prepolymer before UV irradiation. The inclusion of an additional crosslinking agent increases the amount of crosslinking between the reactive prepolymers. Preferable additional crosslinking agents include pentaerythritol tetraacrylate. Additionally, crosslinking agents can be [2+2] cyclized with acrylate based reactive prepolymers to form hydrogels. Thus, while acrylate based reactive prepolymers will not cyclize with themselves, they can be cyclized into hydrogels through the addition of an additional crosslinking agent.

[0074] Preferably, hydrogels have a ratio of cyclized or crosslinked monomers to first monomers of between about one cyclized [2+2] photocyclizable monomer or compound to about every one first monomer and about one cyclized [2+2] photocyclizable monomer or compound to about every 50 first monomers. More preferably, hydrogels have a ratio of cyclized or crosslinked monomers to first monomers of between about one cyclized [2+2] photocyclizable monomer or compound to about every one first monomer and about one cyclized [2+2] photocyclizable monomer or compound to about every 30 first monomers. Most preferably, hydrogels have a ratio of about one cyclized or crosslinked [2+2] photocyclizable monomer or compound to about every 15 first monomers.

[0075] Of the total [2+2] photocyclizable monomers or compounds present in the reactive prepolymer, preferably between 10% and 80% are crosslinked to form the hydrogel, more preferably between 20% and 65% are crosslinked to form the hydrogel, and most preferably between 30% and 50% are crosslinked to form the hydrogel. The remaining uncrosslinked [2+2] photocyclizable monomers or compounds are available for cyclization with probes, solid supports, or other molecules. The approximate percent of crosslinked [2+2] photocyclizable monomers or compounds may be determined by FTIR.

[0076] Solid Supports

[0077] The “solid support” is any surface, including glass, silicon, modified silicon, ceramic, plastic, or polymer, such as (poly)tetrafluoroethylene, or (poly)vinylidenedifluoride, upon which the reactive prepolymer may be placed and attached. Preferable methods of spreading the reactive prepolymer on the solid support include, but are not limited to, roller coating, curtain coating, extrusion coating, offset printing, and spin coating. Most preferably, attachment is by a [2+2] cycloaddition of the reactive prepolymer to the solid support. A preferred solid support is glass.

[0078] The solid support can be any shape or size, and can exist as a separate entity or as an integral part of any apparatus (e.g., bead, curvette, plate, or vessel). The solid support may inherently provide an attachment surface for the reactive prepolymer, or the solid support may be modified to provide adherence of polyacrylamide to the solid support. If the solid support is glass, it is preferably modified with with γ-methacryl-oxypropyl-trimethoxysilane (“Bind Silane,” Pharmacia).

[0079] More preferably, covalent linkage of the polyacrylamide hydrogel to the solid support is performed as described in European Patent Application 0 226 470 or through [2+2] cyclization. The solid support may optionally contain electronic circuitry useful in the detection of bit molecules, or microfluidics used in the transport of micromolecules.

[0080] The preceding description is not intended to limit the scope of the invention to the preferred embodiments described, but rather to enable any person skilled in the art of chemistry to make and use the invention.

EXAMPLES

[0081] Solvents are analytical or HPLC grade. General reagents can be purchased from a variety of commercial suppliers (e.g., Fluka, Aldrich, and Sigma Chemical Co.). Glass slides can be obtained from commercial suppliers (e.g., Corning Glass Works).

Example 1 Method For Synthesizing a 20:1 DMI PRP

[0082] The 20:1 dimethyl maleimide (DMI) based PRP (as depicted in FIG. 1) is a copolymer of acrylamide and bifunctional N-(6-acroloylhexyl)-2,3-dimethyl-maleimide co-monomers. Thus, the PRP is polyacrylamide co-N-(6-acryloylhexyl)-2,3-dimethyl-maleimide.

[0083] First, 17.06 gram (0.24 mol.) of acrylamide (Fluka BioChemica, electrophoresis grade), 3.35 gram (0.012 mol.) of N-(6-acroloylhexyl)-2,3-dimethyl-maleimide, 0.39 gram (0.00156 mol.) of copper(II)sulfate pentahydrate, and 0.3 gram (0.00111 mol.) of potassium peroxodisulfate were dissolved in 81.6 gram of n-propanol/water 2:1 in a 250 mL-3-neck flask equipped with a condenser, a stirrer, and a gas inlet/outlet. The solution was deoxygenated with argon gas for 15 minutes, and then heated to 65° C. and stirred for 4 hours. After cooling to room temperature, the salts were removed from the solution by filtration over a column filled with ion exchange resin (Dowex Monosphere 450).

[0084] The PRP was obtained by adding 0.5 gram anthroquinone 2-sulfonic acid sodium salt as a photosensitiser to 49.5 gram of the above solution recovered from the column. The solid content of this solution was 19.9% by weight. The molecular weight of the PRP was approximately 138,000 on a weight average basis and approximately 3,000 on a number average basis. UV crosslinking of a 5 μm coating of the DMI based PRP followed by imaging and developing operations resulted in formation of a crosslinked polyacrylamide hydrogel array.

Example 2 Method for Synthesizing a 15:1 Glycidyl Acrylate PRP

[0085]15:1 glycidyl acrylate based PRP was formed by using acrylic acid as a second monomer and glycidyl acrylate as a [2+2] photocyclizable compound (as depicted in FIG. 3) or by using glycidyl acrylate as a second monomer and acrylic acid as a [2+2] photocyclizable compound. In the latter case, the polymer backbone was initially a copolymer of acrylamide and glycidyl methacrylate. Thus, the first copolymer formed was polyacrylamide co-glycidyl methacrylate. This first copolymer was further modified with acrylic acid to produce the PRP.

[0086] First, 15.99 gram (0.225 mol.) of acrylamide (Fluka BioChemica, electrophoresis grade), 2.13 gram (0.015 mol.) of glycidyl methacrylate, 0.39 gram (0.00156 mol.) of copper(II)sulfate pentahydrate, and 0.3 gram (0.00111 mol.) of potassium peroxodisulfate were dissolved in 82.5 gram of n-propanole/water 2:1 in a 250 mL-3-neck flask equipped with a condenser, a stirrer, and a gas inlet/outlet. The solution was deoxygenated with argon gas for 15 minutes, and then was heated up to 65° C. and stirred at this temperature for 4 hours. After cooling to room temperature, the salts were removed from the solution by filtration over a column filled with ion exchange resin (Dowex Monosphere 450).

[0087] Forty-five grams of the solution recovered from the column and 0.47 gram (0.0065 mol.) of acrylic acid were combined in a 100 mL flask equipped with a condenser and stirred (magnetic stirrer bar) for 20 hours at 90° C. with external heating. The PRP was obtained after adding 0.45 gram anthroquinone 2-sulfonic acid sodium salt and 0.25 gram triethanol amine to the acrylated acrylamide solution. The solid content of this solution was 23.8% by weight.

Example 3 Photocrosslinking the 20:1 DMI PRP

[0088] A 20% by weight solids aqueous solution (range of from about 2% to about 40% solids) of 20:1 DMI PRP and 1% by weight anthroquinone 2-sulfonic acid sodium salt was coated on a solid support to a wet thickness of about 25 μm (range of from about 2 nanometers to about 5 μm). The coating was then exposed with UV radiation (less than about 1,000 milliJoules/cm²) through a photomask containing a grid array pattern of pads (pad size of from about 1 μm to about 500 μm) to cyclize the exposed PRP into a water insoluble, crosslinked, hydrogel (FIG. 1). Although not shown in FIG. 1, the PRP was simultaneously crosslinked to a glass solid support modified with [2+2] photoreactive sites. The unexposed, and therefore still water-soluble PRP, was then selectively removed by an aqueous developer solution, leaving an array pattern of the crosslinked, porous, hydrogel. Optionally, the solid support was then diced into individual biochips, each containing from about 500 to about 100,000 pads.

Example 4 Photocrosslinking the 15:1 Glycidyl Acrylate PRP

[0089] A 20% by weight solids aqueous solution (range of from 2% to about 40% solids) of 15:1 Glycidyl Acrylate PRP containing 1% by weight pentaerythritol tetraacrylate (range of from about 0.05% to 1.5%) and 0.05% by weight anthroquinone 2-sulfonic acid sodium salt was coated on a solid support to a wet thickness of 25 μm (range of from about 1 μm to about 50 μm). The coating was then exposed with UV radiation through a photomask containing a grid array pattern of pads (pad size of from about 1 μm to about 500 μm) to cyclize the exposed PRP into a water insoluble, crosslinked hydrogel. The unexposed, and therefore still water-soluble PRP was then removed by an aqueous developer solution which left an array pattern of the crosslinked, porous, hydrogel. Optionally, the solid support was diced into individual biochips, each containing from about 500 to about 100,000 hydrogel pads.

Example 5 Forming a Continuous Film Hydrogel

[0090] A 20% by weight solids aqueous solution (range of from about 0.5% to about 40% solids) of 20:1 DMI PRP and 1% by weight anthroquinone 2-sulfonic acid sodium salt was coated on a solid support to a wet thickness of about 25 μm (range of from about 2 nanometers to about 5 μm). The coating was then exposed with UV radiation (less than about 1,000 milliJoules/cm²) to cyclize the exposed PRP into a water insoluble, crosslinked, hydrogel (FIG. 1). Although not shown in FIG. 1, the PRP was simultaneously crosslinked to a glass solid support modified with [2+2] photoreactive sites. The excess, and therefore still water-soluble, PRP was then removed by an aqueous developer solution, leaving a continuous film of the crosslinked, porous, hydrogel.

Example 6 Use of an Additional Crosslinking Agent with an Acrylate PRP and Offset Printing to Form a Pattern.

[0091] A 5% by weight solids aqueous solution (range of from about 2% to about 25% solids) of an acryloyl based polyacrylamide reactive prepolymer containing 1% by weight pentaerythritol tetraacrylate and 1% by weight of an anthroquinone 2-sulfonic acid sodium salt photosensitiser was coated in a grid array pattern (pad size from about 50μm to about 500 μm) by offset printing on a solid support to a wet thickness of 25 μm (range of from about 1 μm to about 50 μm).

[0092] The patterned coating was then exposed with UV radiation to cyclize the exposed PRP into a water insoluble hydrogel, leaving an array pattern of crosslinked, hydrogel material. Optionally, the solid support was diced into individual biochips, each containing from about 500 to about 100,000 hydrogel pads.

Prophetic Example 1 Two-step Synthesis of an Acrylate Based Polyacrylamide Reactive Prepolymer

[0093] A solution of acrylamide and acrylic acid (from about 3% to about 50% acrylic acid) is thermally polymerized between 40 and 50° C. A copolymer of acrylamide and acrylic acid is formed as depicted in FIG. 2. This resultant copolymer is condensed with acryloyl chloride to obtain an acrylate based polyacrylamide reactive prepolymer (PRP) ready for crosslinking. This reaction sequence graphically depicted in FIG. 2.

[0094] As any person skilled in the art of chemistry will recognize from the previous description, figures, and examples that modifications and changes can be made to the preferred embodiments of the invention without departing from the scope of the invention defined by the following claims. 

What is claimed:
 1. A reactive prepolymer comprising a copolymer of a first monomer and a 2+2 photocyclizable monomer.
 2. The reactive prepolymer of claim 1, wherein said copolymer comprises between one and about 50 said first monomers for each said 2+2 photocyclizable monomer.
 3. The reactive prepolymer of claim 1, wherein said copolymer comprises between about 10 and about 20 said first monomers for each said 2+2 photocyclizable monomer.
 4. The reactive prepolymer of claim 1, wherein said reactive prepolymer has the structure:

wherein x is an integer from 1 to 50; y is an integer from 1 to 50; and R is a moiety that comprises a 2+2 photoreactive site.
 5. The reactive prepolymer of claim 4, wherein x is between about 10 and about 20 and y is
 1. 6. The reactive prepolymer of claim 4, wherein x is about 15 and y is
 1. 7. The reactive prepolymer of claim 1, wherein the first monomer is a monomer that is water-soluble and can undergo chain-type polymerization.
 8. The reactive prepolymer of claim 7, wherein the first monomer is selected from the group consisting of acrylamide, hydroxyethyl acrylate, vinyl pyridine, acrylic acid, methacrylic acid, and vinyl pyrrolidone, or mixtures thereof.
 9. The reactive prepolymer of claim 7, wherein the first monomer is acrylamide.
 10. The reactive prepolymer of claim 1, wherein the 2+2 photocyclizable monomer comprises a 2+2 photoreactive site and a polymerizable functionality.
 11. The 2+2 photocyclizable monomer of claim 10, wherein a spacer separates the polymerizable functionality and the 2+2 photoreactive site.
 12. The reactive prepolymer of claim 1, wherein the 2+2 photocyclizable monomer is selected from the group consisting of N-(6-acryloylhexyl)-2,3-dimethylmaleimide and vinyl cinnamate, or mixtures thereof.
 13. The reactive prepolymer of claim 1, wherein the 2+2 photocyclizable monomer comprises a second monomer and a 2+2 photocyclizable compound.
 14. The reactive prepolymer of claim 13, wherein the second monomer is selected from the group consisting of acrylic acid, glycidyl methacrylate, and methacrylic acid, or mixtures thereof.
 15. The reactive prepolymer of claim 13, wherein the second monomer is glycidyl methacrylate.
 16. The reactive prepolymer of claim 13, wherein the second monomer is acrylic acid.
 17. The reactive prepolymer of claim 13, wherein the 2+2 photocyclizable compound is selected from the group consisting of glycidyl methacrylate, acrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, and acryloyl halides, or mixtures thereof.
 18. The reactive prepolymer of claim 15, wherein the 2+2 photocyclizable compound is acrylic acid.
 19. The reactive prepolymer of claim 16, wherein the 2+2 photocyclizable compound is glycidyl methacrylate.
 20. The reactive prepolymer of claim 1, wherein the 2+2 photocyclizable monomer is prepared by forming a copolymer of the first monomer with a second monomer, and subsequently, condensing a 2+2 photocyclizable compound with the copolymer.
 21. The reactive prepolymer of claim 20, wherein the second monomer is selected from the group consisting of acrylic acid, glycidyl methacrylate, and methacrylic acid, or mixtures thereof.
 22. The reactive prepolymer of claim 20, wherein the 2+2 photocyclizable compound is selected from the group consisting of glycidyl methacrylate, acrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, and acryloyl halides, or mixtures thereof.
 23. A hydrogel formed by an ultraviolet irradiation of the reactive prepolymer of claim
 1. 24. A hydrogel formed by an ultraviolet irradiation of the reactive prepolymer of claim
 2. 25. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 8. 26. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 10. 27. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 12. 28. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 13. 29. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 18. 30. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 19. 31. A hydrogel formed by ultraviolet irradiation of the reactive prepolymer of claim
 20. 32. A microarray comprising a solid support and the hydrogel of claim
 23. 33. A microarray comprising a solid support and the hydrogel of claim
 25. 34. A microarray comprising a solid support and the hydrogel of claim
 26. 35. A microarray comprising a solid support and the hydrogel of claim
 27. 36. A microarray comprising a solid support and the hydrogel of claim
 28. 37. A microarray comprising a solid support and the hydrogel of claim
 29. 38. A microarray comprising a solid support and the hydrogel of claim
 30. 39. A microarray comprising a solid support and the hydrogel of claim
 31. 40. A continuous film comprising a solid support and the hydrogel of claim
 23. 41. A continuous film comprising a solid support and the hydrogel of claim
 25. 42. A continuous film comprising a solid support and the hydrogel of claim
 26. 43. A continuous film comprising a solid support and the hydrogel of claim
 27. 44. A continuous film comprising a solid support and the hydrogel of claim
 28. 45. A continuous film comprising a solid support and the hydrogel of claim
 29. 46. A continuous film comprising a solid support and the hydrogel of claim
 30. 47. A continuous film comprising a solid support and the hydrogel of claim
 31. 48. A method of making a reactive prepolymer comprising copolymerizing a first monomer with a 2+2 photocyclizable monomer, wherein the 2+2 photocyclizable monomer comprises a polymerizable functionality and a 2+2 photoreactive site.
 49. A method of making a reactive prepolymer comprising: (a) providing a first monomer; (b) copolymerizing said first monomer with a second monomer; and (c) covalently attaching said second monomer to a 2+2 photocyclizable compound to form a 2+2 photocyclizable monomer.
 50. A method of making a hydrogel comprising: (a) providing a first monomer; (b) providing a 2+2 photocyclizable monomer; (c) polymerizing two or more first monomers with two or more 2+2 photocyclizable monomers, wherein the 2+2 photocyclizable monomers comprise a polymerizable functionality and a 2+2 photoreactive site; and (d) cyclizing at least two of the 2+2 photocyclizable monomers with ultraviolet light to form the hydrogel.
 51. The method of claim 50, wherein said hydrogel has a ratio between about one 2+2 photocyclizable monomer to about every 20 first monomers and about one 2+2 photocyclizable monomer to about every 30 first monomers.
 52. The method of claim 51, wherein between 20% and 65% of said 2+2 photocyclizable monomer is crosslinked.
 53. The method of claim 51, wherein between 30% and 50% of said 2+2 photocyclizable monomer is crosslinked.
 54. The method of claim 50, wherein said hydrogel is between about 2 nanometers and about 5 micrometers in thickness.
 55. The method of claim 50, wherein said hydrogel is between about 2 nanometers and about 100 nanometers in thickness.
 56. The method of claim 50, wherein said hydrogel is cyclized to a solid support.
 57. A method of making a hydrogel comprising: (a) providing a first monomer; (b) providing a second monomer; (c) polymerizing at least two first monomers to at least two second monomers; (d) covalently attaching a 2+2 photocyclizable compound to at least two of the second monomers, wherein said covalent attachment is by a condensation reaction; (e) providing an additional crosslinking agent; and (f) cyclizing at least two of the 2+2 photocyclizable compounds with at least one additional crosslinking agent using ultraviolet light to form the hydrogel.
 58. The method of claim 57, wherein said additional crosslinking agent is pentaerythritol tetraacrylate.
 59. The method of claim 57, wherein said hydrogel has a ratio between about one 2+2 photocyclizable compound to about every 20 first monomers and about one 2+2 photocyclizable compound to about every 30 first monomers.
 60. The method of claim 59, wherein between 20% and 65% of said 2+2 photocyclizable compound is crosslinked.
 61. The method of claim 59, wherein between 30% and 50% of said 2+2 photocyclizable compound is crosslinked.
 62. The method of claim 57, wherein said hydrogel is between about 2 nanometers and about 5 micrometers in thickness.
 63. The method of claim 57, wherein said hydrogel is between about 2 nanometers and about 100 nanometers in thickness.
 64. The method of claim 57, wherein said hydrogel is cyclized to a solid support.
 65. A method of making a hydrogel comprising: (a) providing a first monomer; (b) providing a second monomer; (c) polymerizing at least two first monomers to at least two second monomers; (d) covalently attaching a 2+2 photocyclizable compound to at least two of the second monomers, wherein said covalent attachment is by a condensation reaction; (e) providing the reactive prepolymer of claim 48; and (f) cyclizing at least one of the 2+2 photocyclizable compounds with at least one reactive prepolymer of claim 48 using ultraviolet light to form the hydrogel.
 66. The method of claim 65, wherein said hydrogel has a ratio between about one 2+2 photocyclizable compound to about every 20 first monomers and about one 2+2 photocyclizable compound to about every 30 first monomers.
 67. The method of claim 66, wherein between 30% and 50% of said 2+2 photocyclizable compound is crosslinked.
 68. The method of claim 66, wherein said hydrogel is between about 2 nanometers and about 5 micrometers in thickness.
 69. The method of claim 65, wherein said hydrogel is cyclized to a solid support.
 70. A method of making a hydrogel array comprising: (a) placing a reactive prepolymer including at least two 2+2 photocyclizable sites on a solid support; and (b) cyclizing at least two of the 2+2 photocyclizable sites present in the reactive prepolymer to form a hydrogel array, wherein said hydrogel array is formed by selective irradiation with ultraviolet light.
 71. The method of claim 70, wherein said selective irradiation is with ultraviolet light having a wavelength of about 365 nanometers.
 72. The method of claim 70, wherein said selective irradiation occurs at areas on an array that are not blocked to ultraviolet irradiation by a mask.
 73. The method of claim 70, wherein said selective irradiation occurs at areas on an array irradiated by an ultraviolet laser.
 74. The method of claim 70, wherein a hydrogel location of said hydrogel array is between about 2 nanometers and about 5 micrometers in thickness.
 75. The method of claim 70, wherein a hydrogel location of said hydrogel array is about 200 micrometers in diameter.
 76. The method of claim 70, wherein a hydrogel location of said hydrogel array is about 100 micrometers in diameter.
 77. The method of claim 70, wherein a hydrogel location of said hydrogel array is about 50 micrometers in diameter.
 78. A method of making a continuous hydrogel film comprising: (a) placing a reactive prepolymer including at least two 2+2 photocyclizable sites on a solid support; and (b) cyclizing at least two of the 2+2 photocyclizable sites present in the reactive prepolymer to form a continuous hydrogel film, wherein said continuous hydrogel film is formed by irradiation with ultraviolet light.
 79. The method of claim 78, wherein said irradiation is with ultraviolet light having a wavelength of about 365 nanometers.
 80. The method of claim 78, wherein said continuous hydrogel film is between about 2 nanometers and about 5 micrometers in thickness. 